CN102504013B - Target chemical drug PADM against cancer metastasis and preparation method and use thereof - Google Patents
Target chemical drug PADM against cancer metastasis and preparation method and use thereof Download PDFInfo
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Abstract
The invention discloses a target chemical drug PADM against cancer metastasis and a preparation method and use of PADM. The chemical drug is high-purity PADM hydrochloride with the structure of Ac-Phe-Lys-PABC-ADM.HCl, prepared by purification. The method comprises the following steps of: (1) preparing Fmoc-Lys (MMT), (2) preparing Lys (MMT), (3) preparing Ac-Phe-Lys (MMT), (4) preparing Ac-Phe-Lys (MMT)-PABOH, (5) preparing Ac-Phe-Lys (MMT)-PAB-PNP, (6) preparing Ac-Phe-Lys (MMT)-PABC-ADM, and (7) preparing Ac-Phe-Lys-PABC-ADM.HCl, treating the residue with dichloromethane, and filtering to obtain the final product. The invention also discloses the use of the target chemical drug PADM for preparation of drugs for treating or preventing peritoneal carcinomatosis of gastric cancer. The method is simple and feasible and has stable process and higher yield, and the drug PADM has effective anticancer function.
Description
Technical field
The invention belongs to chemicals field, the present invention relates to the compound PADM (Ac-Phe-Lys-PABC-ADMHCl) of a kind of target Cathepsin B (Cat B) Anti-tumor metastasis, the preparation method who simultaneously relates to this target therapeutic agent, also relate to its application in treatment gastric cancer peritoneum metastatic carcinoma, PADM can efficiently suppress peritonaeum metastatic carcinoma and form in gastric cancer peritoneum metastatic carcinoma nude mice model, and significantly reduce toxic side effect, there is the using value that is developed to treatment gastric cancer peritoneum metastatic carcinoma and other high expression level Cat B malignant tumor medicine.
Background technology
Cancer of the stomach is modal global malignant tumour, especially developing country, and cancer of the stomach incidence and mortality ratio apportion second and the 3rd in male tumor patient, occur in female patient and mortality ratio is all listed as the 4th.In China, gastric cancer mortality is ranked the 3rd, each large tumour.Cancer of the stomach integrated control and the people's life and health is closely bound up.
Early gastric cancer can be treated and must well be treated by radical surgery.Dan China, owing to lacking necessary early screening plan, and public's self-prevention consciousness is not enough, and approximately 80% patients with gastric cancer is being made a definite diagnosis or important organ is being occurred during operations research, and especially intraperitoneal plantation is shifted.Tradition curing gastric cancer method comprises operative treatment, local radiotherapy, adjuvant chemotherapy etc., and these schemes can extend the survival of patients phase to a certain extent.But international cooperating research, comprises that INT-0116 test, MAGIC test and ACTS-GC test all show, surgery alone treatment or the preoperative or postoperative chemicotherapy of operation associating, can not control intraperitoneal relapse and metastasis.It is as effective to cancer of the stomach in the chemotherapy regimen of Zorubicin (Adriamycin, ADM) that MAGIC test shows to contain anthracycline anticancer drug.
Zorubicin is as a kind of important traditional chemotherapeutics, and toxic side effect is serious, mainly comprises cardiac toxic, bone marrow depression, hepatotoxicity etc., and clinical application is seriously limited.Large quantity research shows that the malignant solid tumor Invasion and Metastasis such as cathepsin B (Cathepsin B, Cat B) and cancer of the stomach are closely related both at home and abroad.Cat B has following main characteristic, be mainly distributed in malignant cell endochylema and coating surface, at tumor invasion forward position, content is the highest, and normal tissue cell expression level is extremely low or do not express, and Cat B can the specific modified peptides section of special enzymolysis Phe-Lys simultaneously.The present invention i.e. the characteristic based on Cat B, the synthetic one section of special enzymolysis modification of Cat B base, the formation new drug PADM (Ac-Phe-Lys-PABC-ADMHCl) of adding on ADM molecule.PADM itself is not had a cell killing ability, stable in peripheral blood, to normally or not expressing Cat B cell without (low) toxic side effect; And at tumor tissues especially at tumor invasion forward position, a large amount of special enzymolysis modified peptides of high reactivity Cat B sections discharge a large amount of ADM molecules; ADM is absorbed by tumour cell, thereby realizes the object of target killing tumor cell.
By chemosynthesis, modify ADM molecule and form PADM, it has targeting in Cat B, brings into play anticancer and metastasis of cancer effect, is expected to improve Quality of Life for Patients with Gastric Carcinoma, and Control in recurring shifts, and extends the survival of patients phase.
Summary of the invention
The object of the invention is to be to provide a kind of targeting in the Anti-tumor metastasis chemical drugs PADM (Ac-Phe-Lys-PABC-ADMHCl) of Cat B.PADM is easy to synthesize, and stable existence in blood can be brought into play Efficient killing effect tumour cell Anti-tumor metastasis in tumor tissues, significantly reduces toxic side effect simultaneously.PADM has targeting killing effect to high expression level Cat B malignant tumor tissue, possesses the efficient anti-cancer function of Zorubicin, has avoided the serious cardiac toxic of Zorubicin.
Another object of the present invention is to be to provide a kind of target anticancer to shift the preparation method of chemicals PADM.The method is easy to operate, and conditional stability is easy to production and preparation high purity PADM.Easy to operate, economic and practical, there is good target anticancer ability.
A further object of the present invention is to be to provide a kind of target anticancer to shift the purposes of chemicals PADM in the treatment of gastric cancer peritoneum metastatic carcinoma, PADM can efficiently suppress peritonaeum metastatic carcinoma and form in gastric cancer peritoneum metastatic carcinoma nude mice model, compare with traditional Zorubicin, significantly reduce toxic side effect.
In order to realize above-mentioned object, the present invention adopts following technical measures:
Target anticancer shifts a chemicals PADM, and its concrete molecular formula is C
52h
59n
5o
16.HCl, its concrete structure is Ac-Phe-Lys-PABC-ADMHCl, through purity analysis, obtains high purity PADM hydrochloride.Structure is:
The design of hydrochloride PADM is synthetic, the main high correlation based on Cat B and tumor development, and the characteristic distributions of Cat B in tumor tissues, according to the biological characteristics of the special enzymolysis Phe-Lys of Cat B peptide section, on ADM molecule, the synthetic special enzymolysis modified peptides of Cat B section forms precursor medicine.PADM concrete structure is Ac-Phe-Lys-PABC-ADMHCl; wherein Ac (Acetyl) is ethanoyl, and Phe (Phenylalanyl) is amphetamine acyl group, and Lys (Lysyl) is Methionin acyl group; PABC is to amino carbobenzoxy-(Cbz), and ADM is Zorubicin molecule.PADM is a kind of hydrochloride, can finely be dissolved in water, can stable existence in human serum, containing in Cat B Incubating Solution, can discharge fast ADM (t
1/2=16min).
A kind of target anticancer shifts the preparation method of chemicals PADM.The steps include:
(1) Fmoc-Lys (MMT) fluorenylmethyloxycarbonyl-Methionin (4-methoxyl group trityl group) 1:
In a 500ml tri-neck reaction flasks, add Fmoc-Lys hydrochloride (23.78g, 56.42mmol), anhydrous methylene chloride (250ml), diisopropylethylamine (10.3ml, 1.05eq) and trimethylchlorosilane (15ml, 2.1eq).This mixture is heated to 80 ℃ and refluxes and keep 1 hour, then mixture is cooled to 4 ℃, add thereafter diisopropylethylamine (31ml, 3.1eq) and 4-methoxyl group trityl chloride (18.29g, 1.05eq).Reaction mixture is stirred 15 hours at 25 ℃.After solvent evaporation, by pH=5 buffered soln for residue, water and salt water washing 3 times, then dry with sodium sulphate, filter, after evaporation, obtain yellow powder powder product (34.7g, 96%).
(2) Lys (MMT) Methionin (4-methoxyl group trityl group) 2:
Intermediate Fmoc-Lys (MMT) (5.25g, 8.19mmol), methylene dichloride and the acetonitrile (40ml: 40ml), and then add diethylamine (80ml) that adds the first step in a 250ml round bottom three neck reaction flasks.Reaction mixture stirs 2 hours at 25 ℃, and distillation is except desolventizing, and residue washs with hot acetonitrile, then adds tertiary butyl methyl ether to process.Solid product obtains a mixing solutions with methylene dichloride and dissolve with methanol (1: 1).Remove by filter insolubles, evaporate to dryness filtrate obtains intermediate Lys (MMT) (3.2g, 94%) thereafter.
(3) Ac-Phe-Lys (MMT) ethanoyl-amphetamine acyl group-Methionin (4-methoxyl group trityl group) 3:
In the three neck round-bottomed flasks of a 250ml, add water (25ml) and 1,2-glycol dimethyl ether (70ml).Under nitrogen protection, add previous step reaction product Lys (MMT) (4.09g, 9.78mmol) and lithium hydroxide (410.4mg, 1eq).Then in stirring, add Ac-Phe-OSu (Acetyl-L-Phenylalanyl-N-Oxysuccinimmide, 2.9g, 1.0eq) solution (70ml 1,2-glycol dimethyl ether).Reaction mixture stirs becomes a homogeneous phase solution for 4 hours.Reaction proceeded to after 18 hours, was evaporated to dry.Residue extracts by ethyl acetate, with the washing of pH=4 buffered soln, and then water and salt water washing.The dry concentrated rear light yellow solid product (5.35g, 90%) that produces.
(4) amino (4-methoxyl group trityl group)-p-aminophenyl methyl alcohol 4 of Ac-Phe-Lys (MMT)-PABOH ethanoyl-amphetamine acyl group-Lai:
In a 250ml tri-neck round-bottomed flasks, add Ac-Phe-Lys (MMT) (5.31g, 8.74mmol), methylene dichloride (120ml) and tertiary butyl two carbonic ethers (2.86g, 1.5eq).Under nitrogen protection, add again pyridine (0.74ml, 1.05eq).Reaction mixture stirs after 15 minutes at 25 ℃, adds p-aminophenyl methyl alcohol (1.61g, 1.5eq).Reaction mixture stirred after 18 hours at 25 ℃, and evaporating solvent is to dry.After residue is dry under high vacuum, with tertiary butyl methyl ether, process solid product tertiary butyl methyl ether continuous washing 5 times.Product is 5.24g (84%) after vacuum-drying.
(5) amino (4-methoxyl group the trityl group)-p-aminophenyl methoxyl group-p-nitrophenyl carbonic ether 5 of Ac-Phe-Lys (MMT)-PAB-PNP ethanoyl-amphetamine acyl group-Lai:
Under nitrogen protection, in a 250ml tri-neck round-bottomed flasks, add Ac-Phe-Lys (MMT)-PABOH (5.1g, 7.1mmol), two p-nitrophenyl carbonic ethers (6.5g, 3eq) and fresh molecular sieve (10g).Then at 25 ℃, add again methylene dichloride (120ml) and diisopropylethylamine (3.7ml, 3eq).Reaction mixture stirs 18 hours at 25 ℃, thereafter solids removed by filtration impurity.After filtrate evaporate to dryness, under high vacuum, be dried 6 hours.Thick product can be further purified through recrystallization (methylene dichloride 20ml and tertiary butyl methyl ether 40ml), and solid product after filtration, wash, and vacuum-drying obtains 4.2g (67%).
(6) Ac-Phe-Lys (MMT)-PABC-ADM ethanoyl-amphetamine acyl group-Lai amino (4-methoxyl group trityl group)-to amino carbobenzoxy-(Cbz)-Zorubicin 6:
Under nitrogen protection; in a 250ml tri-neck round-bottomed flasks, add Ac-Phe-Lys (MMT)-PABC-PNP (1.1g; 1.25mmol); Lipodox (763.4mg; 1.05eq); dimethyl formamide (60ml) diisopropylethylamine (0.23ml, 1.05eq).Reaction mixture stirs after 48h at 25 ℃, is adding 400ml ethyl acetate, and organic phase washes with water 4 times, is then evaporated to dry.Thick product obtains orange solid phase prod (0.56g, 60%) through chromatography column purifying (methylene dichloride: methyl alcohol is 20: 1 to 15: 1).Described organic phase: methyl alcohol, acetonitrile, chloroform, hexanaphthene, methylene dichloride, tetrahydrofuran (THF), propyl carbinol, n-propyl alcohol, Virahol, acetone, tetracol phenixin, normal hexane etc.
(7) Ac-Phe-Lys-PABC-ADMHCl PADM 7:
Under nitrogen protection, in a 250ml tri-neck round-bottomed flasks, add 1.89g (1.48mmol) Ac-Phe-Lys (MMT)-PABC-ADM, phenylmethylether (16ml, 100eq) and methylene dichloride (50ml).Reaction mixture adds dichloroacetic acid (1.22ml, 10eq) in stirring at 25 ℃.Reaction mixture is poured in ethyl acetate (400ml) after stirring 2 hours, is then stirring 2 hours, and gained orange solid is after filtering again with ethyl acetate washing 5 times.Solids crude product is dissolved in methyl alcohol (80ml), and gained solution is slowly lived (AG2-X8 ion exchange resin Cl 50g) through ion exchange resin.Contained product component from after be evaporated to dry.Residue is processed and is obtained the finished product by filtration through methylene dichloride.Product obtains 1.51gAc-Phe-Lys-PABC-ADMHCl (98%, HPLC 99.03%Area) after 10 hours in vacuum-drying.Obtain a kind of target anticancer and shifted chemicals PADM.
Target anticancer shifts the application of chemicals PADM in treatment or prevention gastric cancer peritoneum metastatic carcinoma medicine, comprises following content:
(1) the external SGC-7901 stomach cancer cell that kills and wounds: the stomach cancer cell of logarithmic phase, 0.25% (0.25g pancreatin/100mlPBS) trysinization, the RPMI-1640 nutrient solution dilution with containing foetal calf serum, is inoculated in 24 orifice plates, every hole inoculation 1 * 10
5individual cell, after cultivating 48h, (Day 1, D1) get three porocyte countings, establish respectively control group (100 μ l physiological saline), ADM group (0.5 μ g/ml), ADM group (1.0 μ g/ml), PADM group (0.9 μ g/ml), PADM group (1.8 μ g/ml), PADM group (3.6 μ g/ml), continue to cultivate, respectively get cell dissociation every day and with tally counting, continuous 6 days, draw cell growth curve (Fig. 1).Proof PADM has certain lethal effect to tumour cell in vitro.
(2) safe dose is large, and toxic side effect is few: 4 of BALB/c-nude mouses, are divided into dosage A group and B group, 2 every group.With reference to ADM intraperitoneal administration LD
50=13.6 μ g/g.A group, respectively at the 1st, 8,15,22 days, gives 24 μ g/g, 12 μ g/g, 24 μ g/g, 36 μ g/gPADM; B group, respectively at the 1st, 8,15,22 days, gives 36 μ g/g, 18 μ g/g, 36 μ g/g, 54 μ g/g PADM, is 1.5 times (24 μ g/g PADM=13.4 μ g/g ADM) of A group.Every 3 days weigh in (Fig. 2), observes animal state, and sacrificed by exsanguination when animal is dying is cored, liver, the capable histopathological examination of kidney (Fig. 3), shows that PADM safe dose scope is very large, less to the important organ heart, liver, renal toxicity.
(3) SGC-7901 gastric cancer peritoneum cancer model is set up: 6 of BALB/c-nu/nu mouse, 4 subcutaneous injection SGC-7901 cells 5 * 10
6/ 0.2ml, 2 abdominal injection SGC-7901 cells 1 * 10
7/ 0.4ml.Treat that subcutaneous tumors forms, intraperitoneal forms after ascites, and the aseptic tumor tissues that obtains, uses ascites homogenate, forms suspension, and adjusting cell concn is 1 * 10
7/ ml.(Day 0, and DO), D8 is divided into control group (n=9), ADM group (n=10), PADM group (n=10) at random for 29 nude mice abdominal cavity injection homogenate 0.2ml
(4) there is efficient anticancer effect: above-mentioned three experimental group, give physiological saline (10ml/kg), ADM (2mg/kg), PADM (7.2mg/kg) respectively at D8, D12, D16, D20, D24, D28, D32, D36; Tail vein blood 80 μ l before D16, D24, D32 administration, EDTA anti-freezing promoting circulation of blood conventional sense.Within every 2 days, observe animal state, within every 4 days, weigh nude mice body weight.D40 tests termination.Nude mice, dying or research during terminal, is taked sacrificed by exsanguination, 4 ℃ of centrifugation serum after blood coagulation, and part promoting circulation of blood biochemical analysis, remaining-80 ℃ refrigerator storage is standby.Nude mice dissect is checked to peritoneal cancer situation, core, the internal organs such as lung, liver,spleen,kidney, intestines carry out routine pathology and learn and check.Peritoneal cancer metastasis degree represents by experimental peritoneal cancer index (experimental Peritoneal Carcinomatosis Index, ePCI), specific as follows: mouse peritoneal is divided into I district, diaphragm; II district, liver, spleen, stomach ligament and corresponding organs; III district, small intestine, colon and mesentery, stomach wall; IV district, pelvic cavity, urogenital system and rectum.Each district's tumor nodule is marked by following standard: 0 minute, without the visible tubercle of naked eyes; 1 minute, tumor nodule diameter≤2mm; 2 minutes, 2mm < tumor nodule diameter≤5mm; 3 minutes, tumor nodule diameter > 5mm; Bloody ascites 1 minute, without bloody ascites 0 minute; By above-mentioned each district's integration summation, be ePCI.Fig. 4 shows that PADM can reach and even be better than ADM curative effect under current test dose, and can suppress lung and shift.
(5) to zootoxin light side-reaction: body weight change curve (Fig. 5 A), experiment stops nude mice integral status (Fig. 5 B), routine blood test detection of dynamic (table 1), hepatic and renal function index and pathologic finding (Fig. 6, Fig. 7), fully prove that PADM is little on the impact of integral animal situation, avoid bone marrow depression, to liver kidney without overt toxicity.
(6) significantly reduce cardiac toxic: parameters of left ventricular function and pathologic finding (Fig. 8), show that the cardiac toxic of PADM is obviously weaker than ADM, can be good at cardiac function protecting.
The impact of table 1.ADM and PADM human peripheral blood
*P＜0.05，ADM&PADM?vs?Control，at?the?same?time?point；
#P＜0.05，PADM?vs?ADM，at?the?same?time?point.
RBC (red corpuscle), HGB (oxyphorase), PLT (thrombocyte), WBC (white corpuscle), LYM (lymphocyte), NEU (neutrophil leucocyte)
The present invention, by add one section of special enzymatic fragment of Cat B on Zorubicin molecule amino, forms Zorubicin precursor medicine.This novel cpd is no or low toxicity in peripheral blood and normal low expression Cat B tissue, thereby reduced the side effect of Zorubicin; This precursor medicine, in tumor tissue cell, is discharged Zorubicin molecule by active Cat B enzymolysis simultaneously, reaches target killing tumor cell object.
The present invention has following features: (1) PADM is synthetic relatively simple, stable preparation process, and productivity ratio is higher; (2) PADM has efficient anti-cancer function, compares with control group, and peritonaeum tubercle forms obvious minimizing, and ePCI scoring significantly reduces (P=0.003); (3) PADM has significantly reduced the common toxicity of ADM, comprise integrality, PADM treatment group body weight (23.61 ± 0.80) is significantly better than ADM group (18.40 ± 2.97g) (P=0.000), and with control group (24.32 ± 1.40g) indifference, and cardiac toxic, CK and CK-MB all, significantly lower than ADM group (P=0.023 and 0.023), do not have statistical significance etc. and compare difference with normal control.
Accompanying drawing explanation
Fig. 1 is that a kind of PADM is external to SGC-7901 cytosis result schematic diagram.
Show that PADM has certain kill capability to cell in vitro.
Fig. 2 is that a kind of PADM acute toxicity the weight of animals changes schematic diagram.
Prompting PADM is less on the weight of animals and integrality impact.
Fig. 3 is a kind of PADM acute toxicity animal important organ pathology schematic diagram.
A little less than showing that PADM is to the body important organ heart, liver, renal toxicity.A1, A2 and A3 are respectively the low dose group heart, liver, kidney pathological section HE dyeing, and B1, B2 and B3 are respectively the high dose group heart, liver, kidney pathological section HE dyeing, are showed no remarkable damage.
Fig. 4 is that a kind of PADM is to gastric cancer peritoneum metastatic carcinoma curative effect schematic diagram.
Demonstrate the anticancer effect that PADM is good, suitable with conventional medicament Zorubicin under existing dosage level, even more excellent, and can suppress lung and shift.A is ePCI scoring, and PADM and ADM group are significantly lower than control group.B is nude mouse peritonaeum Overall View, and visible PADM and ADM group peritonaeum tubercle all significantly reduce.C is metastasis in control group lung, and PADM and ADM group are showed no transfer case.
Fig. 5 is the whole toxic side effect schematic diagram of a kind of PADM treatment gastric cancer peritoneum metastatic carcinoma.
Show that on the whole PADM is to the obvious weak and traditional Zorubicin of animal side effect.A body weight change curve, ADM group body weight obviously declines, and PADM group is compared body weight and is kept good with control group.B experiment terminal integral animal is seen, and ADM group state is obviously weaker than control group and PADM group.
Fig. 6 is a kind of PADM treatment gastric cancer peritoneum metastatic carcinoma hepatic and renal function biochemical indicator schematic diagram.
Show that PADM is little to hepatic and renal function damage, is better than traditional Zorubicin.
Fig. 7 is that a kind of PADM treatment gastric cancer peritoneum metastatic carcinoma liver-kidney diseases reason checks schematic diagram.
PADM treatment group hepar damnification mild degree, has no obvious Toxicity of Kidney.A1, A2 and A3 are respectively control group, ADM group and PADM group liver organization pathological section HE dyeing.B1, B2 and B3 are respectively control group, ADM group and PADM group renal tissues pathology section HE dyeing.
Fig. 8 is a kind of PADM treatment gastric cancer peritoneum metastatic carcinoma heart function biochemistry and pathologic finding schematic diagram.
The cardiac toxic that fully shows PADM is significantly smaller than traditional Zorubicin.A1, A2 and A3 are respectively three concrete data of important indicator of heart function, and ADM group is all significantly higher than contrast and PADM group.B1, B2 and B3 are respectively control group, ADM group and PADM group typical heart tissue pathological slice HE dyeing.
Embodiment
Embodiment 1:
Target anticancer shifts a preparation method of chemicals PADM, the steps include:
(1) Fmoc-Lys (MMT) fluorenylmethyloxycarbonyl-Methionin (4-methoxyl group trityl group) 1:
In a 500ml tri-neck reaction flasks, add Fmoc-Lys hydrochloride (23.78g, 56.42mmol), anhydrous methylene chloride (250ml), diisopropylethylamine (10.3ml, 1.05eq) and trimethylchlorosilane (15ml, 2.1eq).This mixture is heated to 80 ℃ and refluxes and keep 1 hour, then mixture is cooled to 4 ℃, add thereafter diisopropylethylamine (31ml, 3.1eq) and 4-methoxyl group trityl chloride (18.29g, 1.05eq).Reaction mixture is stirred 15 hours at 25 ℃.After solvent evaporation, by pH=5 buffered soln for residue, water and salt water washing 3 times, then dry with sodium sulphate, filter, after evaporation, obtain yellow powder powder product (34.7g, 96%).
1h-NMR (CDCl3) analytical results: δ 1.26,1.68 (m, 2H, 4H), 2.45 (m, 2H), 3.71 (S, 3H), 4.05-4.40 (m, 4H), 6.81 (d, 2H), 7.15-7.77 (m, 20H).MS(FAB)641(MH)
+，663(M+Na)
+，679(M+K)
+。HRMS?Calc：641.3015，Found：641.3001。
(2) Lys (MMT) Methionin (4-methoxyl group trityl group) 2:
Intermediate Fmoc-Lys (MMT) (5.25g, 8.19mmol), methylene dichloride and the acetonitrile (40ml: 40ml), and then add diethylamine (80ml) that adds the first step in a 250ml round bottom three neck reaction flasks.Reaction mixture stirs 2 hours at 25 ℃, and distillation is except desolventizing, and residue washs with hot acetonitrile, then adds tertiary butyl methyl ether to process.Solid product obtains a mixing solutions with methylene dichloride and dissolve with methanol (1: 1).Remove by filter insolubles, evaporate to dryness filtrate obtains intermediate Lys (MMT) (3.2g, 94%) thereafter.
1h-NMR (DMSO-d6) analytical results: δ 1.34,1.57,1.72 (m, 6H), 2.05 (m, 2H), 3.38 (m, 1H), 3.68 (s, 3H), 3.71 (d, 2H), 7.03-7.40 (m, 12H).MS(FAB)419.2(MH)
+，441.4(M+Na)
+，457.4(M+K)
+。Anal.Calc，C
26H
30N
2O
3·0.5H
2O：C-73.04％，H-7.31％，N-6.55％。Found：C-73.62％，H-7.59，N-6.56％。
(3) Ac-Phe-Lys (MMT) ethanoyl-amphetamine acyl group-Methionin (4-methoxyl group trityl group) 3:
In the three neck round-bottomed flasks of a 250ml, add water (25ml) and 1,2-glycol dimethyl ether (70ml).Under nitrogen protection, add previous step reaction product Lys (MMT) (4.09g, 9.78mmol) and lithium hydroxide (410.4mg, 1eq).Then in stirring, add Ac-Phe-OSu (Acetyl-L-Phenylalanyl-N-Oxysuccinimmide, 2.9g, 1.0eq) solution (70ml 1,2-glycol dimethyl ether).Reaction mixture stirs becomes a homogeneous phase solution for 4 hours.Reaction proceeded to after 18 hours, was evaporated to dry.Residue extracts by ethyl acetate, with the washing of pH=4 buffered soln, and then water and salt water washing.The dry concentrated rear light yellow solid product (5.35g, 90%) that produces.
1h-NMR (CDCl3/CD3OD) analytical results: δ 1.22 (m, 2H), 1.58 (m, 3H), 1.71 (m, 1H), 1.82 (s, 3H), 2.49 (m, 2H), 3.00 (m, 2H), 3.75 (s, 3H), 4.26 (t, 1H), 4.63 (t, 1H), 6.82 (d, 2H), 7.10-7.43 (m, 17H).MS(ESI)608.5(MH)
+。Anal.Calc，C
37H
41N
3O
5·2.5H
2O：C-68.08％，H-7.10％，N-6.44％。Found：C-68.39％，H-7.10％，N-6.23％。
(4) amino (4-methoxyl group trityl group)-p-aminophenyl methyl alcohol 4 of Ac-Phe-Lys (MMT)-PABOH ethanoyl-amphetamine acyl group-Lai:
In a 250ml tri-neck round-bottomed flasks, add Ac-Phe-Lys (MMT) (5.31g, 8.74mmol), methylene dichloride (120ml) and tertiary butyl two carbonic ethers (2.86g, 1.5eq).Under nitrogen protection, add again pyridine (0.74ml, 1.05eq).Reaction mixture stirs after 15 minutes at 25 ℃, adds p-aminophenyl methyl alcohol (1.61g, 1.5eq).Reaction mixture stirred after 18 hours at 25 ℃, and evaporating solvent is to dry.After residue is dry under high vacuum, with tertiary butyl methyl ether, process solid product tertiary butyl methyl ether continuous washing 5 times.Product is 5.24g (84%) after vacuum-drying.
1h-NMR (CDCl3/CD3OD) analytical results: δ 1.31 (m, 1H), 1.50 (m, 1H), 1.89 (m and s, 7H), 2.18 (m, 2H), 3.00 (m, 2H), 3.74 (s, 3H), 4.40 (t, 1H), 4.61 (s, 2H), 4.68 (m, 1H), 6.67 (d, 1H), 6.77 (d, 2H), 7.00-7.55 (m, 21H), 8.92 (br, 1H).MS(ESI)713.6(MH)
+，735.7(M+Na)
+。Anal.Calc?C
44H
48N
4O
5·0.5H
2O：C-73.21％，H-6.84％，N-7.76％。Found：C-73.48％，H-7.07％，N-7.71％。
(5) amino (4-methoxyl group the trityl group)-p-aminophenyl methoxyl group-p-nitrophenyl carbonic ether 5 of Ac-Phe-Lys (MMT)-PAB-PNP ethanoyl-amphetamine acyl group-Lai:
Under nitrogen protection, in a 250ml tri-neck round-bottomed flasks, add Ac-Phe-Lys (MMT)-PABOH (5.1g, 7.1mmol), two p-nitrophenyl carbonic ethers (6.5g, 3eq) and fresh molecular sieve (10g).Then at 25 ℃, add again methylene dichloride (120ml) and diisopropylethylamine (3.7ml, 3eq).Reaction mixture stirs 18 hours at 25 ℃, thereafter solids removed by filtration impurity.After filtrate evaporate to dryness, under high vacuum, be dried 6 hours.Thick product can be further purified through recrystallization (methylene dichloride 20ml and tertiary butyl methyl ether 40ml), and solid product after filtration, wash, and vacuum-drying obtains 4.2g (67%).
1h-NMR (CDCl3/CD3OD) analytical results: δ 1.31 (m, 1H), 1.50 (m, 1H), 1.89 (m and s, 7H), 2.18 (m, 2H), 3.00 (m, 2H), 3.74 (s, 3H), 4.40 (t, 1H), 4.61 (s, 2H), 4.68 (m, 1H), 6.67 (d, 1H), 6.77 (d, 2H), 7.00-7.55 (m, 21H), 8.92 (br, 1H).MS(ESI)713.6(MH)
+，735.7(M+Na)
+。Anal.Calc?C
44H
48N
4O
5·0.5H
2O：C-73.21％，H-6.84％，N-7.76％。Found：C-73.48％，H-7.07％，N-7.71％。
(6) Ac-Phe-Lys (MMT)-PABC-ADM ethanoyl-amphetamine acyl group-Lai amino (4-methoxyl group trityl group)-to amino carbobenzoxy-(Cbz)-Zorubicin 6:
Under nitrogen protection; in a 250ml tri-neck round-bottomed flasks, add Ac-Phe-Lys (MMT)-PABC-PNP (1.1g; 1.25mmol); Lipodox (763.4mg; 1.05eq); dimethyl formamide (60ml) diisopropylethylamine (0.23ml, 1.05eq).Reaction mixture stirs after 48h at 25 ℃, is adding 400ml ethyl acetate, and organic phase washes with water 4 times, is then evaporated to dry.Thick product obtains orange solid phase prod (0.56g, 60%) through chromatography column purifying (methylene dichloride: methyl alcohol is 20: 1 to 15: 1).
1h-NMR (DMF-d7) analytical results: δ 1.25 (d, 3H), 1.41 (m, 2H), 1.56 (m, 2H), 1.87 (m and s, 4H), 2.09 (m, 4H), 2.34 (m, 4H), 3.12 (m, 4H), 3.63 (brs, 1H), 3.78 (s, 3H), 3.92 (m, 1H), 4.11 (s, 3H), 4.33 (m, 1H), 4.51 (m, 1H), 4.68 (m, 1H), 4.81 (s, 2H), 4.90 (m, 1H), 5.00 (brs, 2H), 5.13 (brs, 1H), 5.40 (brs, 1H), 5.61 (s, 1H), 6.78 (d, 1H), 6.89 (d, 2H), 7.28 (m, 17H), 7.50 (d, 4H), 7.71 (m, 3H), 8.05 (m, 3H), 9.98 (s, 1H).MS(ESI)1280.3(M-H)
-，1282.4(MH)
+。Anal.Calc?C
72H
75N
5O
17·2H
2O：C-65.59％，H-6.04％，N-5.31％。Found：C-65.46％，H-5.99％，N-5.25％。
(7) Ac-Phe-Lys-PABC-ADMHCl PADM 7:
Under nitrogen protection, in a 250ml tri-neck round-bottomed flasks, add 1.89g (1.48mmol) Ac-Phe-Lys (MMT)-PABC-ADM, phenylmethylether (16ml, 100eq) and methylene dichloride (50ml).Reaction mixture adds dichloroacetic acid (1.22ml, 10eq) in stirring at 25 ℃.Reaction mixture is poured in ethyl acetate (400ml) after stirring 2 hours, is then stirring 2 hours, and gained orange solid is after filtering again with ethyl acetate washing 5 times.Solids crude product is dissolved in methyl alcohol (80ml), and gained solution is slowly lived (AG2-X8 ion exchange resin Cl 50g) through ion exchange resin.Contained product component from after be evaporated to dry.Residue is processed and is obtained the finished product by filtration through methylene dichloride.Product obtains 1.51gAc-Phe-Lys-PABC-ADMHCl (98%, HPLC 99.03%Area) after 10 hours in vacuum-drying.Obtain a kind of target anticancer and shifted chemicals PADM.
1h-NMR (DMF-d7) analytical results: δ 1.21 (d, 3H), 1.52 (m, 2H), 1.80 (s and m, 9H), 2.22 (m, 1H), 2.39 (m, 1H), 3.03 (m, 6H), 3.60 (m, 1H), 3.87 (m, 1H), 4.05 (s, 3H), 4.30 (m, 1H), 4.52 (m, 1H), 4.67 (m, 1H), 4.74 (s, 2H), 4.91 (m, 2H), 5.11 (brs, 1H), 5.38 (brs, 1H), 5.60 (brs, 1H), 6.75 (d, 1H), 7.26 (m, 7H), 7.71 (m, 3H), 7.91 (m, 1H), 8.32 (d, 1H), 8.45 (br, 3H), 10.21 (brs, 1H).MS(ESI)1010.5(MH)
+。Anal.calc?C
52H
60N
5O
16Cl·2.5H
2O：C-57.22％，H-6.00％，N-6.41％。Found：C-57.16％，H-6.03％，N-6.34％.
The test of embodiment 2:PADM in the medicine of preparation treatment or prevention gastric cancer peritoneum metastatic carcinoma.
A kind of target anticancer shifts the application of chemicals PADM in preparation treatment or prevention gastric cancer peritoneum metastatic carcinoma medicine.Comprise following content: In vitro cell experiment, toxicity in vivo experiment and gastric cancer peritoneum metastatic carcinoma animal model experiment.
(1) In vitro cell experiment:
The external SGC-7901 stomach cancer cell that kills and wounds: the stomach cancer cell of logarithmic phase, 0.25% (0.25g pancreatin/100ml PBS) trysinization, the RPMI-1640 nutrient solution dilution with containing foetal calf serum, is inoculated in 24 orifice plates, every hole inoculation 1 * 10
5individual cell, after cultivating 48h, (Day 1, D1) get three porocyte countings, establish respectively control group (100 μ l physiological saline), ADM group (0.5 μ g/ml), ADM group (1.0 μ g/ml), PADM group (0.9 μ g/ml), PADM group (1.8 μ g/ml), PADM group (3.6 μ g/ml), continue to cultivate, respectively get cell dissociation every day and with tally counting, continuous 6 days, draw cell growth curve (Fig. 1).
(2) toxicity in vivo experiment:
4 of BALB/c-nude mouses, are divided into dosage A group and B group, 2 every group.With reference to ADM intraperitoneal administration LD
50=13.6 μ g/g.A group, respectively at the 1st, 8,15,22 days, gives 24 μ g/g, 12 μ g/g, 24 μ g/g, 36 μ g/g PADM; B group, respectively at the 1st, 8,15,22 days, gives 36 μ g/g, 18 μ g/g, 36 μ g/g, 54 μ g/g PADM, is 1.5 times (24 μ g/g PADM=13.4 μ g/g ADM) of A group.Every 3 days weigh in (Fig. 2), observes animal state, and sacrificed by exsanguination when animal is dying is cored, liver, the capable histopathological examination of kidney (Fig. 3).
(3) gastric cancer peritoneum metastatic carcinoma animal model experiment:
6 of A.BALB/c-nu/nu mouse, 4 subcutaneous injection SGC-7901 cells 5 * 10
6/ 0.2ml, 2 abdominal injection SGC-7901 cells 1 * 10
7/ 0.4ml.Treat that subcutaneous tumors forms, intraperitoneal forms after ascites, and the aseptic tumor tissues that obtains, uses ascites homogenate, forms suspension, and adjusting cell concn is 1 * 10
7/ ml.(Day 0, and DO), D8 is divided into control group (n=9), ADM group (n=10), PADM group (n=10) at random, sets up gastric cancer peritoneum metastatic carcinoma model for 29 nude mice abdominal cavity injection homogenate 0.2ml.
B. control group, ADM group and PADM group, give physiological saline (10ml/kg), ADM (2mg/kg), PADM (7.2mg/kg) respectively at D8, D12, D16, D20, D24, D28, D32, D36; Tail vein blood 80 μ l before D16, D24, D32 administration, EDTA anti-freezing promoting circulation of blood conventional sense.Within every 2 days, observe animal state, within every 4 days, weigh nude mice body weight.D40 tests termination.Nude mice, dying or research during terminal, is taked sacrificed by exsanguination, 4 ℃ of centrifugation serum after blood coagulation, and part promoting circulation of blood biochemical analysis, remaining-80 ℃ refrigerator storage is standby.Nude mice dissect is checked to peritoneal cancer situation, core, the internal organs such as lung, liver,spleen,kidney, intestines carry out routine pathology and learn and check.Peritoneal cancer metastasis degree represents by experimental peritoneal cancer index (experimental Peritoneal Carcinomatosis Index, ePCI), specific as follows: mouse peritoneal is divided into I district, diaphragm; II district, liver, spleen, stomach ligament and corresponding organs; III district, small intestine, colon and mesentery, stomach wall; IV district, pelvic cavity, urogenital system and rectum.Each district's tumor nodule is marked by following standard: 0 minute, without the visible tubercle of naked eyes; 1 minute, tumor nodule diameter≤2mm; 2 minutes, 2mm < tumor nodule diameter≤5mm; 3 minutes, tumor nodule diameter > 5mm; Bloody ascites 1 minute, without bloody ascites 0 minute; By above-mentioned each district's integration summation, be ePCI (Fig. 4).
C. evaluating toxic and side effect.Integral animal state estimation comprises that body weight change curve (Fig. 5 A) and experiment stop nude mice integral status (Fig. 5 B).Routine blood test detection of dynamic (table 1), hepatic and renal function index and pathologic finding (Fig. 6,7), parameters of left ventricular function and pathologic finding (Fig. 8).
Claims (1)
1. target anticancer shifts the application of chemicals PADM in preparation treatment gastric cancer peritoneum metastatic carcinoma medicine, and described PADM is:
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| WO1998013059A1 (en) * | 1996-09-27 | 1998-04-02 | Bristol-Myers Squibb Company | Hydrolyzable prodrugs for delivery of anticancer drugs to metastatic cells |
| CN101234204A (en) * | 2008-02-28 | 2008-08-06 | 中国科学院长春应用化学研究所 | High molecule bonding adriamycin medicine, nano capsule and preparation thereof |
| CN101259284A (en) * | 2008-04-15 | 2008-09-10 | 华东师范大学 | Liver target anticancer nano prodrug system based on tree shaped polymer, preparation and use |
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| WO1998013059A1 (en) * | 1996-09-27 | 1998-04-02 | Bristol-Myers Squibb Company | Hydrolyzable prodrugs for delivery of anticancer drugs to metastatic cells |
| CN101234204A (en) * | 2008-02-28 | 2008-08-06 | 中国科学院长春应用化学研究所 | High molecule bonding adriamycin medicine, nano capsule and preparation thereof |
| CN101259284A (en) * | 2008-04-15 | 2008-09-10 | 华东师范大学 | Liver target anticancer nano prodrug system based on tree shaped polymer, preparation and use |
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